Showing papers by "Christopher M. Overall published in 1988"

Identification and characterization of enamel proteinases isolated from developing enamel. Amelogeninolytic serine proteinases are associated with enamel maturation in pig.

Abstract: During tooth formation nearly all of the protein matrix of enamel is removed before final mineralization. To study this process, enamel proteins and proteinases were extracted from pig enamel at different stages of tooth development. In the enamel maturation zones, the major enamel matrix proteins, the amelogenins, were rapidly processed and removed. Possibly associated with this process in vivo are two groups of proteinases which were identified in the enamel extracts by enzymography using amelogenin-substrate and gelatin-substrate polyacrylamide gels and by the degradation in vitro of guanidinium chloride-extracted amelogenins. One group of proteinases with gelatinolytic activity consisted of several neutral metalloendoproteinases having Mr values from 62,000 to 130,000. These proteinases were inactive against amelogenins, casein and albumin, and were present in approximately equal proportions in enamel at all developmental stages. In the other group, two serine proteinases, with apparent non-reduced Mr of 31,000 and 36,000 exhibited amelogeninolytic activity. The substrate preference of the enamel serine proteinases was indicated by their limited degradation of casein and their inability to degrade gelatin and albumin. Contrasting with the distribution of the metalloendoproteinase enzymes, the serine proteinases were found only in the enamel scrapings taken from late-maturing enamel. The amelogenin degradation patterns in vivo, observed in the enamel scrapings, were similar to those produced in assays in vitro using partially purified fractions of enamel proteinases and amelogenin substrate. Together, these data strongly indicate an important role for the serine proteinases, and possibly the gelatinolytic proteinases, in the organized processing of the enamel protein matrix during enamel formation.

Identification of matrix metalloendoproteinase inhibitor (TIMP) in human parotid and submandibular saliva: partial purification and characterization.

Abstract: Matrix metalloendoproteinase inhibitor (TIMP) is constitutively expressed by a variety of cells and is present in most connective tissues, and in serum and amniotic fluid. In our previous studies, collagenase inhibitor activity has been identified in saliva (5). To determine the nature of this inhibitor, fresh human parotid saliva was first concentrated by ammonium sulfate precipitation and the 20–60% saturated ammonium sulfate fraction containing the inhibitor activity was chromatographed on an AcA 54 gel filtration column. The inhibitor eluted with an apparent Mr of 29000 and a 42-fold purification was achieved. For characterization, the inhibitor was further purified by heparin-Sepharose chromatography. The inhibitor, which bound to heparin-Sepharose in 0.0 M NaCl at neutral pH and remained bound after a 0.18 M NaCl step elution, was eluted with 0.25 M NaCl. The partially purified inhibitor was characterized by its stability at low pH (pH 2.0) and after heat treatment (60° C and 95° C), and by its resistance to organomercurial (APMA) and trypsin treatments. These properties, together with immunoreactivity with an anti-human TIMP polyclonal antibody on immunoblots. are consistent with the collagenase inhibitor activity being TIMP. Collagenase in either the active or precursor forms was not delected in parotid or submandibular ductal saliva nor after AcA 54 chromatography of parotid ductal saliva. The presence of TIMP in saliva has important implications. First, it has the potential to suppress the activity of matrix metalloendoproteinases released during periodontal inflammation; and second, in the analysis of collagenolytic enzymes in gingival crevicular fluid, it is clear that care must be taken to avoid contamination of the crevicular fluid with saliva.

Biochemical comparison of fibroblast populations from different periodontal tissues: characterization of matrix protein and collagenolytic enzyme synthesis.

Abstract: To compare the expression of extracellular matrix components by fibroblasts from different periodontal tissues, rat molar periodontal ligament fibroblasts (RPL) and rat gingival fibroblasts (RGF) were isolated and cultured from individual animals. Pulse–chase experiments using [35S]methionine as a precursor revealed that confluent populations of early passage cells of both cell types synthesized similar amounts of collagen, fibronectin, and SPARC/osteonectin. Qualitative and quantitative differences were apparent in the relative proportions of type III collagen, in the rates of procollagen processing, and in the synthesis of a small number of unidentified proteins observed by sodium dodecyl sulphate – polyacrylamide gel electrophoresis. Collagen constituted 24–26% of the radiolabelled proteins secreted by both cell types, type I being the predominant collagen, with lower amounts of type III (3–8% RGF, 8–18% RPL) and type V (~1%) collagens. Procollagen processing in the culture medium of RPL cells was more...